Diagnosis, prevention, and/or treatment of atherosclerosis and underlying and/or related diseases

ABSTRACT

Complement is recognized as an important, humoral defense system involved in the innate (nonspecific) recognition and elimination of microbial invaders, other foreign particles or molecules, and antigen-antibody complexes from the body. The present invention makes use of the surprising notion that the handling of lipids by the body, rather than its antimicrobial activity, is the primary and most ancient function of the complement system. Consequently, atherosclerosis as observed in disorders associated with disturbed lipid metabolism (familial combined hyperlipidemia (FCHL), postprandial hyperlipidemia, hypertriglyceridemia with low levels of HDL cholesterol, and insulin resistance associated with type-II diabetes and obesity), is ascribed to either genetic or acquired defects in ancient (activatory and/or regulatory) complement components. Based on this new insight, novel preventive measures and treatment modalities of disturbed lipid metabolism are introduced.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/327,604 filed Dec. 20, 2002, pending, which application is acontinuation-in-part of PCT International Patent ApplicationPCT/NL01/00673, filed on Sep. 12, 2001, designating the United States ofAmerica, and published, in English, as WO 02/22161 A2 on Mar. 21, 2002,the contents of the entirety of each of which is incorporated herein bythis reference. This application also claims benefit under 35 U.S.C. §119(e) to U.S. Provisional Patent Application Ser. No. 60/253,465 filedon Nov. 28, 2000.

TECHNICAL FIELD

The invention relates to the diagnosis, prevention, and/or treatment ofatherosclerosis and/or underlying and/or associated diseases.

BACKGROUND

According to the classical view, atherosclerosis is a conditionultimately leading to the narrowing of blood vessels, impairedcirculation, and restricted oxygenation of tissues.⁽¹⁾ If this processoccurs in heart vessels (coronary arteries), consequences are theclinical conditions of angina pectoris and myocardial infarction; in thebrain, atherosclerosis leads to cerebrovascular accidents; in the legs,the clinical presentation is claudicatio intennittens. Classical riskfactors Associated with atherosclerosis are: obesity, hypertension,smoking, diabetes, male gender, fasting hyperlipidemia, and especiallyincreased cholesterol concentrations. Novel risk factors have emergedduring the last decennia, these including hyperhomocysteinemia,hypertriglyceridemia with low HDL cholesterol concentrations,postprandial hyperlipidemia, the insulin resistance syndrome, and apositive family history for cardiovascular disease, among others.

According to epidemiological surveys, coronary heart disease (CHD) isthe leading cause of death in western societies. In the United Kingdomin 1987, 31% of all deaths in males (280,177 total deaths in men) and24% in females (total number: 286,817) were due to CHD. More than onequarter of CHD deaths in men (total CHD mortality in men: 86,978)occurred before the age of 65 years. In women (68,257 CHD deaths), thevast majority (almost 75%) occurred at ages beyond 75 years. The Dutchsituation is similar and representative for other countries in Westernsociety. In The Netherlands in 1997, there were 135,783 deaths in total(67,242 males and 68,541 females). In men, 37% of total mortality(24,664 deaths) was due to CHD and in women 38% (25,881 deaths). From1972 to 1997, mortality due to CHD in The Netherlands decreased by 44%(age-corrected); however, hospital admissions related to CHD increasedby 53%. This decrease in CHD-associated mortality is probably ascribedto improved care in coronary-care and intensive-care units. In addition,the early recognition of the above-mentioned risk factors for CHD andimproved treatment of these risk factors may have led to increasedsurvival in patients at risk.

The classical drugs for the treatment of these risk factors arecholesterol-lowering drugs (mainly statins),⁽³⁾ drugs aiming at thereduction of blood pressure like angiotensin-converting-enzymeinhibitors⁽⁴⁾ and drugs like aspirin which act on clot formation. Theeffects of lifestyle change to reduce body weight and stopping smokinghave been disappointing so far, although their impact has not beenestablished adequately on a population basis. Improvement of regulationof diabetes has resulted in decreased morbidity (less amputations, lessdiabetics with end-stage renal failure necessitating dialysis, and lessdiabetics becoming blind),^((5, 6)) but the incidence of cardiovasculardisease in diabetics did not decrease by these measures.^((6, 7))

Many investigators point at the need for the recognition of concealedrisk factors for CHD in diabetes (and obesity) and a more aggressivetreatment of these factors should result in improved outcome. Moreover,landmark trials with lipid-lowering drugs in secondary and primaryprevention settings have resulted in significantly decreased mortalityin treated patients (30% risk reduction),⁽²⁾ but there were stillsignificant numbers of patients that could not be saved by these drugs.Therefore, the identification of additional risk factors and thedevelopment of novel therapeutic interventions are expected to result ina significant reduction of total mortality due to CHD.

It has been postulated that atherosclerosis is associated with animpaired clearance of chylomicron remnants, i.e., partially hydrolyzedchylomicrons (intestinally derived triglyceride-rich lipoproteins).Also, it was suggested (PCT International Patent Publication WO00/34469) that clusterin could be used as a migration inhibitor ofvascular smooth muscle cells in arteries whose migration andproliferation may lead to vessel injury and arterial lesion and whosemigration and proliferation can be induced by atherosclerosis. Rosenbergand Silkensen,⁽¹⁰⁾ in reviewing the multifunctional protein role ofclusterin state the determination of a common mechanism underlying itsvarious functions would lead to a key in comprehending an important areaof biology. Other researchers⁽¹¹⁾ have demonstrated that clusterin (apoJ) may have a protective role against atherosclerosis as it participatesin cholesterol transport.

One of the recently recognized mechanisms in the development ofatherosclerosis is inflammation.⁽⁸⁾ Several studies have demonstratedthat slightly elevated concentrations of C-reactive protein (CRP; awell-known acute-phase reactant named after its reactivity with theso-called C-polysaccharide of pneumococci) are predictive of coronaryevents in middle-aged and elderly men and women. However, the precisemechanism by which complement is involved in atherosclerosis is notknown. In discussing a possible relationship between infections withpathogenic micro-organisms, MBL (an innate immune-defense plasmaprotein) deficiency and atherosclerosis, Madsen et al.⁽¹²⁾ suggested thepresence of unexpected non-infective mechanisms relevant to thedevelopment of atherosclerosis but could not conclusively exclude arelationship with other pathogens. The role of MBL in the immune systemand the use of recombinant MBL in treating deficiencies in the immunesystem is well known (PCT International Patent Publication WO 00/70043).The present invention teaches how lipid metabolism, complementactivation, atherogenic processes and immune responses arephysiologically related.

SUMMARY OF THE INVENTION

The present inventors have elucidated a mechanism providing anexplanation for the insufficient protective effects of lipid-loweringdrugs, the persistently high incidence of coronary heart disease inwestern societies, and the relationship with markers of inflammationlike CRP. As a result of this new insight, a novel approach for thediagnosis, prevention, and/or therapy of atherosclerosis and underlyingor related disease(s) is presented which comprises a method for thetreatment and/or prophylaxis of diseases associated with disturbances inthe complement/lipid pathway by modulating the activity of one or moreelements in the pathway. Such a new method may for instance beimplemented at a large scale in combination with current strategies tolower mortality and morbidity by CHD.

The present invention makes use of the surprising notion that theprimary and most ancient function of the complement system is thetransport and targeting of lipoproteins (i.e., chylomicrons, VLDLs,LDLs, and their remnants) to the liver, rather than its antimicrobialactivity. Consequently, atherosclerosis as observed in disordersassociated with disturbed lipid metabolism (familial combinedhyperlipemia (FCHL), postprandial hyperlipidemia, hypertriglyceridemiawith low levels of HDL cholesterol, and insulin resistance associatedwith type-II diabetes and obesity), must be ascribed to either geneticor acquired defects in ancient (activatory and/or regulatory) complementcomponents. Based on this new insight, novel preventive measures andtreatment modalities of disturbed lipid metabolism are introduced.

In accordance with the invention, it has surprisingly been found thatclearance of chylomicron remnants and in general clearance of alltriglyceride-rich particles (chylomicrons, VLDL, IDL and their remnants)and LDL particles is positively regulated by the complement system; thatis to say by the most ancient complement activation pathways, the“lectin” and “alternative” pathways. Delayed clearance oftriglyceride-rich particles, in particular those containingapolipoprotein B as a structural protein, is related to deficiencies inthe ancient complement activation pathways. Moreover, in one embodimentthe invention predicts that low serum levels of the intercellular matrixproteins vitronectin and/or clusterin, which function as regulators ofthe “terminal” or “lytic” pathways of complement, lead to decreasedintravascular integrity of chylomicron remnants. Such a decreasedintegrity is typically atherogenic.

Accordingly, the invention relates to the use of purified or enrichedphysiologic complement components, physiologic complement regulatorsand/or extrinsic complement modulators of natural (e.g., plant-derived),synthetic, or semi-synthetic origin in the prevention and/or treatmentof atherosclerosis and underlying and/or related diseases bysubstituting for and/or at least diminishing deficiencies in thecomplement activation pathways.

Because a thorough and mechanistic insight has now been achieved, theinvention provides novel diagnostic tools and formulations of specificand highly effective primary and secondary prevention strategies fordisturbances leading to atherosclerosis. Dependent on what is (are) theweakest link(s) in the specific pathways of the complement system in anindividual patient, a physician can, based on the considerations of theinvention, modulate the activity of the complement system of the patientin order to prevent and/or treat manifestations of disease.

The present invention has as an objective to provide new and improvedmanners of prevention and/or treatment of atherosclerosis andunderlying/related diseases. The invention further provides new andimproved manners of determining the occurrence (diagnosis) ofatherosclerosis and related diseases, in particular those which areassociated with disturbed lipid metabolism and to classify thesediseases accordingly.

A further object of the present invention is to provide for coordinateddesign and discovery of new drugs for the treatment of atherosclerosisand related diseases as well as providing compositions comprisingmodulators of the complement activation pathways which can serve as abasis or an ingredient of a pharmaceutical composition or a foodproduct. The present invention therefore also relates to pharmaceuticalproducts or food products that comprise such modulating compositions. Asa further object, the present invention provides the use of at least onecomplement factor or modulator for the manufacture of a medicament forthe treatment and/or prevention of atherosclerosis or an underlyingand/or related disease.

In order to appreciate the importance of the invention, the inventorsdeem it necessary to explain the newly developed concept in much moredetail. The surprisingly intricate relationship between the complementsystem and the clearance of chylomicron remnants unraveled by theinventors signifies a pathway not hitherto known. This unexpectedfinding gives rise to measures for treatment and prophylaxis ofatherosclerosis that are themselves surprising, and that lead to theidentification of additional risk factors and the development of noveltherapeutic interventions which results in a significant reduction oftotal mortality due to CHD.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the complement system in schematic representation.

FIG. 2 shows the two most ancient pathways of the complement system in aschematic representation.

FIG. 3 shows the relationship between triglyceride-rich particles (TRP),their remnants (TRP-R), the position of lipoprotein lipase (LPL), freefatty-acids (FFA), acylation-stimulating protein (ASP), severalcomplement components (C3, factor B and factor D) in relation totriglyceride (TG) uptake by adipocytes and liver-derived very lowdensity lipoproteins (VLDL).

FIGS. 4A to E show the binding to chylomicrons of C3, MBL, clusterin andvitronectin as determined in Example 1.

FIGS. 5A and B show the effect of immune adherence of triglyceride-richparticles to erythrocytes in blood after staining with Sudan Black asdetermined in Example 2.

FIG. 6 shows the flow cytogram obtained after staining apo B on humanerythrocytes as determined in Example 3.

FIG. 7 shows the internalization of triglyceride-rich particles in ablood leukocyte as determined in Example 4.

FIG. 8 shows the complement/lipid pathway in schematic representation.

FIG. 9 shows the potency of different substances to activate complementin vitro.

FIG. 10 shows the effects of glycosylated plant stanols on fastingtriglycerides, plasma apoB and plasma cholesterol levels over a fourmonth period in a patient with heterozygous FamilialHypercholesterolemia.

FIG. 11 a shows the effect of vitamin A on postprandial C3 plasmaconcentration after two hours in healthy lean volunteers.

FIG. 11 b shows the effect of vitamin A on postprandial plasmatriglyceride concentration after two hours in healthy lean volunteers.

DETAILED DESCRIPTION OF THE INVENTION The Complement System GeneralDescription

The complement system⁽⁹⁾ is a complex signaling system comprisingenzymes present in blood. The complement system is involved in the earlyrecognition and clearance of foreign bodies and antigen-antibodycomplexes (also called immune complexes) from the circulation andtissues. Complement is recognized as an important, humoral defensesystem involved in the innate (nonspecific) recognition and eliminationof microbial invaders, other foreign particles or molecules, andantigen-antibody complexes from the body.

Upon the recognition of foreign material in tissue or blood, the mostcrucial and abundant complement component, C3, is activated by C3convertases. This activation triggers a cascade of events thatultimately leads to the clearance of the foreign material. C3,consisting of an a chain and a β chain, is activated through asplit-conversion into C3b and C3a (FIG. 1). C3a represents theN-terminus (77 amino acids) of the α chain and C3b represents theC-termini of the α and β chains. C3 convertases, of which various formsexist, can be generated through three different complement activationpathways (FIG. 1) and its synthesis is well regulated. TheC3-convertase-generating pathways include, in order of descendingevolutionary age, the so-called “lectin” pathway (LP), the “alternative”pathway (AP) which is also known as the “amplification loop,” and therelatively young “classical” pathway (CP). During evolution, anadditional system known as “terminal” or “lytic” pathway has developedon top of the complement activation system, which can destabilizemembranes of, e.g., Gram-negative bacteria, virus-infected body cells,or even tumor cells by pore formation, resulting in their killing.Phylogenetic studies have pointed out that the “lectin” and“alternative” pathways are by far the most ancient complement activationpathways (about 700 million years; FIG. 2), whereas the “classical” and“lytic” pathways are relatively young (400 to 350 million years).

The complex nature of the complement system can be appreciated whenfollowing the fate of the split-products of C3. One of the splitproducts, C3a, is a spasminogen and anaphylatoxin, which induces therelease of histamine from basophilic cells, including tissue mast cellsand basophilic granulocytes. Histamine, in turn, helps phagocytes toleave the blood vessels in order to arrive at the site of complementactivation, i.e., the accumulation site of foreign material or immunecomplex. In blood, C3a is rapidly (in about 15 minutes) inactivated byserum carboxypeptidases. The most prominent serum carboxypeptidase (sCP)in blood is the constitutively expressed sCP-N. All other sCP types areinducible and are less abundant than the N type. Upon the inactivationof C3a by carboxypeptidases, the C-terminal arginine is removed,resulting in the generation of C3adesArg. This compound is (probablyidentical to) an acylation-stimulating protein (ASP), a hormone that canstimulate fat accumulation in the body.

C3b and its inactivated form C3bi are opsonins, which means that theycan bind covalently to sugar OH groups (via ester bonds) or protein NH₂groups (via amide bonds) on material identified as “foreign.” In case ofsuch binding events, foreign material is also termed “substrate.” Othercomplement components can also function as opsonins, among these areother C3-derivatives and the complement component C4 (see below) andderivatives thereof. Opsonins promote the clearance of foreign materialby the blood-based monocytes and tissue-based macrophages, both known asmononuclear phagocytes. The mononuclear phagocytic system is present inthe liver, spleen, lymph nodes, or the affected tissue itself. Thesespecialized cells carry specific complement receptors on their surfacethat can bind the opsonins. Known complement receptors on phagocytes areCR1, CR3, and possibly also CR4. CR1 is an exclusive receptor of C3bwhereas CR3 and CR4 are also able to bind C3bi. In contrast tomononuclear phagocytes, polymorphonuclear phagocytes (PMNs) arerelatively inefficient in eliminating foreign material, at least in theabsence of antibodies.

In primates, immune complexes are eliminated by the mononuclearphagocytic system in liver, spleen and bone after erythrocyte-mediatedtransport via the blood stream. The erythrocytes carry a restrictednumber of CR1 molecules on their surface to which C3b(i)-coated immunecomplexes can adhere. This phenomenon is called “immune adherence.”Erythrocytes of non-primate species are CR1 negative and consequently donot mediate the transport of immune complexes to liver, spleen and bone.In primates suffering from systemic autoimmune diseases and neoplasticdiseases (cancer), the clearance of immune complexes involvesantibody-mediated activation of the complement system.

Microbial pathogens in the circulation are also cleared by themononuclear phagocytic system, but only after MBL (“lectin”pathway)-mediated or antibody/C1 (“classical” pathway)-mediatedactivation of complement components C4, C2, and C3. This process isknown to involve erythrocyte-mediated clearance as well.

The phenomenon of “immune adherence,” as it has turned out, is one ofimportance to the present invention, as the present inventors have foundthat these CR1 complement receptors do not only bind immune complexes ormicrobial pathogens, but also chylomicrons and other triglyceride-richparticles and their remnants. Based on this finding, new methods for thetreatment and prophylaxis of atherosclerosis and related disease haveemerged that are based on intervention or modulation of the complementpathways involved.

As mentioned, the complement system comprises several pathways each witha multitude of protein compounds, signaling molecules, receptors,regulators and activators. To appreciate the scope of the presentinvention, the various pathways of complement activation will bedescribed in some more detail.

The Complement System: The “Lectin” Pathway

Activation of the “lectin” pathway (LP) starts with the recognition andbinding of foreign bodies by a serum lectin, called mannose-bindinglectin (MBL). MBL is a high-molecular-weight, sugar-binding protein,present in minute amounts (about 2 μg per ml) in blood plasma. MBL andthe lung surfactant proteins A (LspA) and D (LspD), belongs to thefamily of the collagenous lectins (collectins). C1, the first componentin the “classical” pathway is a collectin-like activator of C4 and C2.Upon binding of MBL to foreign bodies, a number of MBL-associatedproteins (MASPs—which are themselves esterases) become coordinatelyactivated, ultimately leading to the generation of the active forms ofthe associated proteins, the LP-dependent C4, C2 and/or C3 convertases.These convertases, which have C3, C4 and C2 as their natural substrates,generate essentially five products: C3b and C3a, C4bC2a and splitproducts C4a and C2b. Like C3b, the C4b portion of C4bC2a bindscovalently to its substrate (e.g., polysaccharides or (glyco)proteins onbacteria) via ester or amide bonds, and is therefore known as anopsonin. The two split products, C4a and C2b, are released in the fluidphase. Substrate-bound C4bC2a is the LP-dependent C3 convertase, causingthe conversion of C3 into C3b and C3a. Like C3a, C4a is a spasminogenand anaphylatoxin (histamine liberator), whereas C2b has kinin-likeactivity. Furthermore, one of the MBL-associated proteins is capable ofdirect activation of C3.

MBL recognizes foreign bodies by its six identical sugar-bindingmoieties with specificity for mannose, N-acetyl-glucosamine, and fucose.This makes sense, because microbial pathogens like fungi, yeasts, and,e.g., Mycobacteria carry relatively high amounts of mannose, whilepeptidoglycan of gram-positive bacteria contains N-acetyl-glucosamine asone of its major building blocks.

The Complement System: The “Alternative” Pathway

Until the discovery of the “lectin” pathway in 1989, the “alternative”pathway (AP, also known as alternate pathway or alternative complementpathway), first described in 1956, was considered the most ancientcomplement activation route. The main function of this “alternative”pathway is to increase (amplify) the number of C3-converting sites onthe substrate of complement activation: the foreign body or the immunecomplex. This means that, once “non-self” material has been identifiedby MBL and activation of the “lectin” pathway has consequently takenplace, the LP-dependent C3 convertase C4bC2a present on the substratewill be amplified by AP-dependent C3 convertases in the following manner(FIG. 1): Substrate-bound C3b, generated by the LP-dependent C3convertase C4bC2a, will bind AP component factor B which, in turn, willbe activated to Bb by AP component factor D (also known as adipsin) toform the AP-dependent C3 convertase (C3bBb). Along with the formation ofthis new C3 convertase, the factor-B part loses a split product calledBa. The enzymic function of the AP-dependent C3 convertase isconsiderably stabilized upon the binding of AP component “properdin”(factor P), resulting in the AP-dependent C3 convertase complex C3bBbP.Split product Ba is a leukotaxin, which helps to direct the movement ofphagocytes to the site of complement activation (primary inflammationsite).

The net result of AP activation is an increase in the number of C3b andinactivated C3b (C3bi) moieties on the substrate, which promote therecognition and clearance of foreign bodies and immune complexes by,predominantly, mononuclear phagocytes (monocytes/macrophages).

The Complement System: The “Classical” Pathway

The “classical” pathway (CP) is generally considered the youngestcomplement activation route, since it is dependent on antibodies (IgMand IgG), which appeared relatively late in phylogeny (from about 350million years ago). The CP is very similar to, and therefore probablyderived from the ancient “lectin” pathway, since the first CP component(C1; consisting of a complex of the collectin-like C1q and two MASP-likeproteins called C1r and C1s) is both phenotypically and functionallyvery much related to the MBL/MASPs complex. In addition, the “classical”pathway involves “lectin” pathway complement components C4 and C2. Likethe sugar-bound MBL/MASPs complex, C1 (composed of C1q, C1r, and C1s)bound to IgM- or IgG-type immune complexes becomes coordinatelyactivated to form a C1-esterase which has C4 and C2 as its naturalsubstrates and which gives rise to the generation of CP-dependent C3convertases, which are identical to LP-dependent C3 convertases(substrate-bound C4bC2a complexes).

C4 exists in two isoforms known as C4A and C4B. C4A is involved in theclearance phenomenon, whereas C4B is mainly involved in the killing ofbacteria and cell destruction (e.g., hemolysis). In the presentdescription, C4 is understood to relate to the C4A isoform unlessotherwise stated.

The Complement System: The Terminal or “Lytic” Pathway

When a newly formed C3b molecule does not bind to the substratedirectly, but to another, substrate-bound C3 convertase (C4bC2a orC3bBbP), triple or quadruple complexes consisting of C4bC2aC3b orC3bBbC3bP are formed. These complexes have C5-converting activityindicating that they are able to split complement component C5 into C5band C5a. This is the starting point of the so-called “terminal” or“lytic” complement pathway. Like Ba, C5a is a leukotaxin, but morepotent than Ba. C5b forms a complex with C6 and C7, the resultant ofwhich is a soluble C5b-7 complex, which has affinity for membranousbilayers. Upon insertion into a membrane of, e.g., a gram-negativebacterium, complement component C8 will bind to the complex, whichresults in a new enzyme, the membrane-bound C9 polymerase (C5b-8). Underthe influence of one C5b-8 complex, some 13 C9 molecules becomepolymerized, resulting in a cylindrical pore in the membrane that isunder attack. Depending on the total number of membrane-bound poly-C9pores, and on whether the bacterium is encapsulated or not, theGram-negative bacterium will either be killed or be able to resist andsurvive membrane attack.

The Complement System: Complement Regulation and Complement Regulators

In order to prevent unwanted activation of the complement cascade, e.g.,by cells of the body itself (homologous cells, in contrast to foreign orheterologous cells), complement activation on homologous cells isheavily regulated by both cell-bound complement inhibitors andregulators in the fluid phase (e.g., serum or plasma).

The most important soluble regulators are:

-   -   For the “lectin” pathway: α₂-Macroglobulin (α2M), serpines and        C4-binding protein (C4BP), which interfere with the formation of        the LP-dependent C4/C2-convertase (activated MBL/MASPs complex)        and the subsequent activation of C4 and C2;    -   For the “alternative” pathway: Factor H (also known as β1H) and        factor H-like molecules, acting at the level of factor B binding        to target-bound C3b (preventing the formation of AP-dependent C3        convertases), C3b inactivator (factor I), acting in conjunction        with factor H, to convert C3b in its enzymatically inactive, but        as opsonin still active form C3bi;    -   For the “classical” pathway: C1INH, an inhibitor of complement        component C1, acting at the level of activated C1, the        C1-esterase (C1INH is also an inhibitor of other serine        esterases such as kallikrein, the clotting factors XIa and XIIa,        and the fibrinolysis product plasmin); and    -   For the “lytic” pathway: Vitronectin (S protein) and clusterin        (also known as apolipoprotein J or apo J). These proteins act at        the level of C5b-7 complexes, preventing their insertion into        bilayer membranes and inhibiting C9 polymerization and        consequently, the lysis of bacteria, viruses and body cells.

Cell-bound complement regulators include:

-   -   Complement receptor 1 (CR1) which has factor-H-like co-enzyme        function versus factor I; CR1 is present on phagocytes,        platelets, but also as a carrier protein on erythrocytes;    -   Decay-accelerating factor (DAF, which is also known as cluster        of differentiation protein CD55) and membrane cofactor protein        (MCP=CD46), both acting at the level of AP activation;    -   Homologous restriction factor with 20-k molecular mass        (HRF20=CD59) and HRF60, both inhibitory at the level of C9        polymerase (C5b-8) formation; and    -   Sialic acid, which acts similar to CD55 and CD46 at the level of        the AP-dependent C3-convertase formation, but also on C9        polymerization.

The Complement System: Complement Activation and the Innate and SpecificImmune System

Apart from the physiological activatory and regulatory complementcomponents mentioned above, different substances of bacterial, plant,animal, or (semi)synthetic origin are known to either activate orinhibit the complement cascade(s). These components include, i.e.,bacterial lipopolysaccharides, β-glycyrrhetinic acid, phytosterols,bovine conglutinin, and polymeric substances like dextran sulphate andglucans.

Bacterial lipopolysaccharides have recently been recognized as potentactivators of the “lectin” pathway. Likewise, β-glycyrrhetinic acid, asa possible activator of C4, was suggested to be able to activate the“lectin” pathway, while the phytosterols with as most importantrepesentatives β-sitosterol, stigmasterol, and campesterol, have beenshown to activate the “alternative” pathway. Dextran sulphate functionsas an acceptor site for “alternative” pathway regulatory protein factorH, and thus facilitates the “alternative” pathway-mediated activation ofC3 and subsequent deposition of C3b on a substrate.

Based on their complement-activating capacity, a number of thesesubstances, including bacterial lipopolysaccharides, dextran sulphates,and glucans, as well as lipidated muramyl-dipeptides and lipophilicquaternary ammonium compounds like dimethyldioctadecyl ammonium bromide,show potent immunological adjuvant activity, which means that they areable to stimulate antigen-specific T- and B-cell responses.

Lipid Metabolism the Physiology of Lipid Metabolism

Under physiologic conditions, about 90% of the ingested fat(triglycerides) is taken up by the epithelial cells of the smallintestine, resulting in the generation of intestinally derivedtriglyceride-rich lipoproteins, called chylomicrons. These chylomicronsare transcytosed through the epithelial cells and delivered at theirbasolateral side to the sub-epithelial interstitium. The structure ofchylomicrons is stabilized by a large, highly glycosylated protein,called apolipoprotein B48 (apo B48), of which the most dominant glucosesresidues are: mannose (17.8%), N-acetyl-glucosamine (16.8%), galactose(13.4%), and fucose (3.4%) which, in fact, fully matches with thebinding specificities of MBL. Apo B48 is the 5′ splice product of alarger apob gene, which, in human intestinal epithelial cells, ispost-transcriptionally modified by a unique editing enzyme. Thismodification results in a premature stop codon leading to thetranslation of only 48% of the apob mRNA. Since the human liver lacksthe unique editing enzyme, apob transcription in the liver results inthe synthesis of full-length apo B100. This protein is the structuralprotein of the liver-derived triglyceride-rich particles known as VLDL(very low density lipoproteins) and their remnants (IDLs and LDLs).

From the sub-epithelial interstitium, chylomicrons are collected intissue fluid (lymph). Via lymph vessels, they are transported tosubsequent draining lymph nodes and, through the thoracic duct and theleft subclavian vein, they finally arrive in the blood stream. Once inthe circulation, chylomicrons are rapidly converted into chylomicronremnants by the action of vascular-endothelium-associated lipoproteinlipase (LPL). Chylomicron remnants are present in blood in differentsizes.

Chylomicrons and chylomicron remnants are subsequently clearedefficiently by the liver from where they can undergo bile-mediatedexcretion via the stools. However, the efficiency of the process ofchylomicron and chylomicron-remnant targeting to the liver is far fromunderstood, while the subsequent hepatic clearance of thesetriglyceride-rich particles has not completely been elucidated either.In the liver, it involves at least the activity of the hepatictriglyceride lipase (HTGL), interaction with specific apo E receptors,and non-receptor binding to the cellular surface in the hepatic space ofDisse. Several local receptors may be involved includinglow-density-lipoprotein receptor-related protein/α₂-Macroglobulinreceptor (LRP-α₂M), a parenchymal liver cell “chylomicron remnantreceptor,” the asialoglycoprotein receptor, the lipolysis-stimulatedreceptor, and the IDL (low density lipoprotein) receptor. Recently, theVLDL receptor, a new member of the LDL receptor supergene family, whichis not present in the liver, has been recognized as a physiologicalreceptor for chylomicron remnants.

Cholesterol, delivered to the liver by chylomicrons and chylomicronremnants, is largely re-secreted into the circulation afterincorporation into very-low density lipoproteins (VLDL). Thischolesterol is further employed by the adrenals and genitals as askeleton for their steroid-hormone synthesis.

Free fatty acids (FFA) arising from the breakdown of chylomicrons by theendothelial LPL are transported over the mucosa towards sub-endothelialfat cells (adipocytes) in which they become re-esterified intointracellular triglycerides (FIG. 3). The uptake and incorporation ofFFA into adipocytes is under the positive control of a hormone calledacylation-stimulating protein (ASP).

Similarly to the hydrolysis of triglycerides in chylomicrons, VLDL maybecome VLDL remnants also called IDL (intermediate-density lipoproteins)by the lipolytic action of LPL, in this case under the positive andnegative control of two other apolipoproteins, apo CII and apo cIII,⁽¹¹⁾respectively. IDL are rich in apo E which functions as the ligand forthe hepatic LDL receptor and “remnant-receptor” (=LRP,LDL-receptor-related protein, a member of the LDL-receptor familycomprising complement repeats; possibly older than the LDL-receptoritself). Apo E (formerly “Arginine-Rich Apoprotein”) is one of theprotein constituents of triglyceride-rich lipoproteins. Chylomicronremnants depend on apo E for their binding to the receptors, since theapo B48 structural protein does not contain the (carboxy-terminal)binding site for the LDL-receptor and “remnant receptor.” Apo E issynthesized by almost all tissues but not by the epithelium of theintestine. The major organ responsible for apo E synthesis is the liver.As a result, chylomicrons receive apo E from HDL in the circulation and,therefore, apo E is an exchangeable apoprotein. In the liver-sinusoids,hepatocytes secrete apo E resulting in an enrichment of remnantparticles, thereby facilitating their removal from the circulation.There are three major apo E isoforms which are genetically determined:Apo E3 (the most common), apo E2 (which results in a minority of thecases in dysbetalipoproteinemia in homozygotes), and apo E4. The latterhas the highest affinity for binding to the receptors, while apo E2exhibits the lowest affinity. Apo E4-individuals are highly responsiveto dietary changes and cholesterol and fat enriched diets lead to higherplasma cholesterol concentrations in these individuals, due todown-regulation of LDL-receptors.

Under physiological conditions, IDL are taken up by LDL-receptors in theliver, by which organ the lipoproteins are degraded and cholesterol isremoved from the body by excretion into the bile.

Although much is known, the metabolic pathways of the intestinally andliver-derived triglyceride-rich particles in blood, chylomicrons andVLDL, respectively, and their remnants have hitherto only partially beenidentified. It has been shown that these pathways comprise commonelements and show a certain overlap. However, until the presentinvention, the very efficient targeting to the liver of chylomicrons andchylomicron remnants under physiological conditions and their clearancewas far from understood.

Lipid Metabolism Aberrant Lipid and/or Free-Fatty-Acid Metabolism

Chylomicron remnants are potentially atherogenic (atherosclerosisgenerating) particles due to their ability to directly induce foam-cellformation, without any modification. Low-density lipoprotein particles(LDL), in contrast, must be oxidized before they induce transformationof mononuclear phagocytes into foam cells. Mononuclear phagocytes havean LDL receptor by which they are able to bind, take up, internalize andsubsequently degrade native LDL. As soon as the intracellular freecholesterol levels reach a threshold value the IDL receptors aredown-regulated and the internalization process is stopped. Oxidized LDLparticles, on the other hand, are taken up by “scavenger” receptors,which are not down-regulated by cholesterol.

Since chylomicrons, VLDL, and their remnants compete for the samemetabolic pathways, patients with delayed remnant clearance mayexperience a temporary accumulation of chylomicrons and chylomicronremnants in the circulation, which obviously contributes to the processof atherogenesis. Such situations are likely to occur in patients withfamilial combined hyperlipidemia (FCHL), type-2 diabetes mellitus,insulin resistance, and obesity. Enhanced plasma VLDL levels in thesesituations are associated with delayed clearance of chylomicronremnants.

Similar mechanisms are involved in conditions in which the clearance ofremnant particles is impaired due to mutations in the apo E ligand gene(type III hyperlipidemia=familial dysbetalipoproteinemia), the LDLreceptor (familial hypercholesterolemia; FH), familial defective apoB100 (FDB) and after menopause. In these conditions, which are allassociated with the development of (premature) atherosclerosis, adelayed clearance of chylomicron remnants has been established due to animpaired binding to receptors in the liver. Other disorders associatedwith impaired remnant clearance are apo CII deficiency, (partial)lipoprotein lipase (LPL) deficiency, and hepatic triglyceride lipase(HTGL) deficiency. In these disorders, the conversion oftriglyceride-rich particles into their remnants is delayed, leading toan accumulation in the circulation of triglyceride-rich particles ofdifferent sizes and triglyceride content.

In many endocrinological disorders like hypothyroidism, growth hormonedeficiency, hypercortisolism by endogenous or exogenous corticosteroids,and the postmenopausal state, a decreased clearance of chylomicronremnants has been established when compared with the control situation.Finally, in patients with premature atherosclerosis and normal fastingplasma lipids (40% of all patients with myocardial infarction below 60years of age in males and beyond 65 years of age in females),chylomicron-remnant clearance is decreased. It has been hypothesized andit is widely accepted that this may be one of the important mechanismsunderlying atherosclerosis in these groups of patients. Identificationof the underlying defect(s) in these patients and modulation andimprovement of their chylomicron-remnant clearance will contribute to areduction of the risk for coronary artery disease and therefore todecreased morbidity and mortality.

Free fatty acids (FFA) arising from the breakdown of chylomicrons by theendothelial lipoprotein lipase (LPL) and their uptake by the adipocytesstimulate these adipocytes to synthesize complement component C3 and“alternative” pathway components factors B and D (note that in healthyindividuals, there is a linear relationship between total body fat andC3 levels), and according to the invention complement activation occurs.

The prior art discloses several important pathways involved in lipidmetabolism and remnant clearance. However, designing optimal treatmentand/or prophylactic measures for atherosclerosis and underlying and/orrelated diseases have thus far been impossible to achieve. It is nowfound by the present inventors that the existence of a pathway that washitherto unknown allows for the first time the development of suchmeasures based on a more complete and physiological and immunologicalunderstanding of the diseases. The surprisingly intricate relationshipbetween the complement system and the clearance of chylomicron remnantsunraveled by the inventors signifies the presence of such a pathway,which is termed the lipid eliminating complement activation pathway orcomplement/lipid pathway.

Due to this new finding the identification of additional risk factors,novel therapeutic interventions and pharmaceuticals and the treatmentand prophylaxis of atherosclerosis have now become available, which willresult in a significant reduction in occurrence and/or progression ofthis disease and other diseases associated with this pathway. The novelpathway was revealed inter alia by three independent findings. The firstfinding comprises that chylomicrons can induce complement activation.The second finding comprises that chylomicrons bind to erythrocyteswhich binding comprises complement factors as a result of which lipidtransport through the blood is complement and erythrocyte mediated. Thethird finding relates to the glycosylation of apolipoprotein B, itskinship to MBL binding specificity and the insight that thecomplement-mediated lipid transport may thus be modulated throughintervention in the complement/lipid pathway and its individual elementsor components. Such elements or components are understood to compriseall molecules and complex substances that play a role in thecomplement/lipid pathway.

It has now surprisingly been found that chylomicrons, isolated fromhealthy individuals after an oral fat load, carry complement componentsC3 (i.e., the opsonins C3b and/or C3bi) (FIG. 4A). Thus, thesechylomicrons initiate complement activation. In addition, it was alsosurprisingly found that chylomicrons, isolated from healthy individualsafter an oral fat load, also carry the “lectin” pathway complementcomponent mannose-binding lectin (MBL), and theterminal-complement-pathway inhibitors clusterin and vitronectin (FIGS.4B-E). Thus, chylomicrons activate the “lectin” pathway (MBL-binding)which may ultimately lead to opsonization with C3b(i) (see “lectin”pathway)) and to binding to the CR1 receptor of phagocytes anderythrocytes (see general description of complement system).Furthermore, the presence of clusterin and vitronectin indicates acapacity to inhibit the “terminal” pathway of the human complementsystem.

It was indeed found that virtually all erythrocytes of healthyvolunteers carry chylomicrons and chylomicron remnants (FIG. 5A),whereas erythrocytes in the “fasting” state carry considerably lesschylomicrons and chylomicron remnants (FIG. 5B). This finding is inaccordance with the new concept of an erythrocyte-mediated eliminationof triglyceride-rich particles (and possibly also LDL particles) andcomplement-mediated lipid transport, and can be interpreted in terms ofimmune adherence of remnant particles and targeting of lipids to theliver and spleen.

The pathway revealed by the present inventors provides an explanationfor the observed complement activation and for a more completephysiological and immunological understanding of atherosclerosis and/orunderlying and/or related disease. The present inventors disclose thatthe prominent glycosylation sites of apolipoproteins B48 and B100, thatare present as structural proteins on plasma chylomicrons and VLDL,respectively, match fully with the mannose, N-acetylglucosamine, and/orfucose binding specificity of MBL. This means that triglyceride-richparticles (LDL, chylomicrons, VLDL, etc.) in blood directly activate thecomplement system's “lectin” pathway through binding of apolipoprotein Bto MBL.

As an intrinsic complement activator (of MBL), apo B is potentially veryharmful (note the existence of autoantibodies against the C3 convertasesF-42 and C3 nephritic factor in patients with collagen diseases). Inparticular, the intrinsic complement activatory nature of the structuralapolipoprotein B molecules of triglyceride-rich particles is nowpredicted to be harmful for individuals with decreased serum levels of“terminal” pathway inhibitors vitronectin and/or clusterin, since such asituation will, subsequent to “lectin” pathway activation, allow“terminal” pathway activation to occur. “Terminal” pathway activation ontriglyceride-rich particles may result in the release of atherogeniclipid material, particularly in patients with a genetic or acquireddeficiency in the “terminal” pathway regulators vitronectin orclusterin. The binding of the “terminal” pathway inhibitors vitronectinand clusterin to chylomicrons can teleologically be explained in termsof protection from atherosclerosis.

Combination of chylomicron (remnant)-induced complement activation ofthe “lectin” pathway, the matching of glycosylation sites ofapolipoproteins B48 and B100, and the erythrocyte-mediated eliminationof triglyceride-rich particles predicts that increased levels oftriglyceride-rich particles in blood, as occurring in FCHL and otherdisorders associated with atherogenic disturbances of lipid metabolism,is due to sub optimal erythrocyte-dependent clearance of chylomicronsand/or VLDL.

Also, disturbances in chylomicron- and/or VLDL- and/orchylomicron-remnant- and/or VLDL-remnant-mediated complement activationwill lead to impaired lipid metabolism. Likewise, disturbances in thecomplement cascade, albeit subtle and, e.g., acquired, may also lead toimpaired lipid metabolism and, in the long term, to atherosclerosis andCHD.

This bears considerable consequences for the treatment and prophylaxisof all diseases related to the complement/lipid pathway, specificallythose relating to disturbances in lipid metabolism. Such diseases arerecognized to comprise atherosclerosis and underlying or relateddisorders which include, but are not limited to, ischemia,hyperlipidemia, such as familial combined hyperlipemia (FCHL),postprandial hyperlipidemia and hypertriglyceridemia with low levels ofHDL cholesterol, insulin resistance associated with type-II diabetes,obesity, coronary heart disease and premature atherosclerosis.

Other diseases related to the disturbances in the complement/lipidpathway are more immunological in appearance. The similarity in theirelimination pathways predicts that triglyceride-rich particles have tocompete with soluble immune complexes and/or microbes for eliminationsites on erythrocytes and in the liver and spleen, which would explainthe disturbed lipid metabolism in, e.g., septic shock. This bearsconsiderable consequences for the treatment and prophylaxis of diseasessuch as, but not limited to, the auto-immune disorders systemic lupuserythematosus (SLE), rheumatoid arthritis (RA) and paroxysmal nocturnalhemoglobinuria (PNH), virtually all infectious diseases and relateddisorders such as AIDS-related (secondary) lipodystrophy, septic shock,and multiple organ failure, inflammatory diseases such as Crohn'sdisease, inflammatory bowel syndrome (IBS), thermal injury includingburns and frostbite, uveitis, psoriasis, asthma and neoplastic diseasessuch as cancer. This immunological aspect of the present invention holdsconsequences for improving the effectiveness of vaccination programs.

Disorders directly related to the complement/lipid pathway comprise:

-   -   disturbances in chylomicron-, chylomicron-remnant, VLDL- and/or        VLDL-remnant-mediated complement activation,    -   disturbances in the complement cascade itself,    -   disturbances in erythrocyte-dependent chylomicron remnant and/or        VLDL-remnant clearance,    -   disturbances in the complement-mediated lipid metabolism,        disturbances in the regulation of lipid metabolism.

Such disorders are atherogenic and may lead to atherosclerosis and/or anunderlying and/or related disease or to a disease directly related todisturbed lipid metabolism or to a disease which may seem to be morerelated to an immunological disorder or malfunction such as auto-immunediseases, infectious diseases, neoplastic diseases and/or inflammatorydiseases.

Treatment and/or prophylaxis can as a benefit of the present inventionoccur by correction of disturbed complement function, in case ofimpaired complement-mediated lipid metabolism and will lead to anamelioration of lipid metabolism. By correcting the disturbed complementfunction, in case of impaired complement-mediated lipid metabolism, anamelioration of disorders associated with impaired or disturbedchylomicron remnant clearance is achieved.

Further, correction of disturbed complement function, in case ofimpaired complement-mediated lipid metabolism, will result in anamelioration of atherosclerosis and underlying or otherwise relateddiseases such as FCHL, insulin resistance in association with type-2diabetes and/or obesity, or coronary heart disease/prematureatherosclerosis.

Further, correction of disturbed complement function, in case ofimpaired complement-mediated lipid metabolism, will result in anamelioration of diseases of the immune system, as well as concomitantinfectious, autoimmune, neoplastic or hematological diseases related toimpaired complement-dependent lipid metabolism.

Disturbances of lipid metabolism due to delayed or disturbederythrocyte-dependent clearance of chylomicrons and/or VLDL may have anumber of possible causes, which will determine the nature of thecorrective measure. There may be:

-   -   (i) congenital defects in glycosylation of apo B48 and/or apo        B100; or    -   (ii) absolute (homozygous), or relative or acquired deficiencies        of individual complement components of the “lectin” and        “alternative” pathways (such deficiencies are known to occur for        MBL (9% of the population), C4A (defective gene frequency 10 to        13% of the population), C4B (defective gene frequency 7 to 18%        of the population), C2 (rare), C3 (rare), factor B (rare) and        factor D (rare)); or    -   (iii) deficiencies of serum carboxypeptidases (sCP) which        exclude the conversion of C3a into C3adesArg (incidence        unknown); or    -   (iv) absolute (rare) or relative (quite common) deficiencies of        complement receptor 1 (CR1) on erythrocytes as occurring in some        patients with systemic lupus erythematosus (SLE);    -   (v) deficiencies of terminal-pathway regulator vitronectin (4%        of the population), which may lead to the lysis of        triglyceride-rich particles resulting in unwanted deposition of        lipids; or    -   (vi) decreased serum levels of clusterin in association with        exacerbations of SLE or with circulating immune complexes        accompanying neoplastic diseases (deficiencies of clusterin are        rare; <<1% of the population).

The incidence of serious cardiovascular disease (37% in 1997) in theNetherlands expressed as percentage of total numbers of fatal cases peryear, matches well with the combined figures for MBL, C4A, C4B,vitronectin, and clusterin deficiencies, corrected for the incidence ofdouble and triple deficiencies.

It is one embodiment of the present invention to provide a method forthe treatment and/or prophylaxis of diseases associated withdisturbances in the complement/lipid pathway by modulating the activityof one or more elements in the pathway.

In another embodiment according to the invention the activity of one ormore elements of the lectin pathway and/or the alternative pathway ofcomplement activation are modulated.

Modulating according to the present invention should be understood asregulating, controlling, blocking, inhibiting, stimulating, activating,mimicking, bypassing, correcting, removing, washing, administering,adding, and/or substituting one or more elements in the pathway or, inmore general terms, intervening in the pathway.

In one aspect of the invention, the elements in this pathway comprisetriglyceride-rich particles and/or their remnants and their constitutiveproteins, complement proteins, complement activators, complementinhibitors, complement regulators and/or complement receptors.

In one embodiment, the activity of one or more elements is modulatedthrough administration of a modulator.

Modulators according to the invention are substances that can bringabout a modulation in the complement/lipid pathway or the complementsystem and may comprise triglyceride-rich particles and/or remnantsthereof and/or constitutive proteins thereof, complement proteins,complement activators, complement inhibitors such as serpines, factor H,factor I and/or C1INH, complement regulators such as α2M, theirmetabolic precursors, encoding genes and/or fragments thereof and theymay be of physiologic (human or primate-derived), natural (e.g.,plant-derived), recombinant, synthetic and/or semi-synthetic origin inenriched, purified and/or chemically modified, complete and/or partialform, as metabolic precursor, as biochemically functional analogue or asfunctional equivalent of a (physiologic) modulator and/or derivativesthereof used alone or in combination.

“Functional equivalents” as used herein are understood to comprisemolecules having at least one function of the original compound,preferably all functions of the original compound (although notnecessarily to the same extent), more preferably chemically similarcompounds, most preferably compounds differing by at most three groupsnot relevant for the relevant activity and/or function of the originalcompound. In the context of the present invention, functionalequivalents of complement factors are understood to comprise the splitproducts of these factors.

In a preferred embodiment, modulators may be MBL-replacement factors,which exhibit one or more functions of the mannose binding lectin suchas binding to C3b or a mimetic thereof, and/or binding to the prominentapo B glycosylation sites or mimetics thereof. Such an MBL-replacementfactor may comprise lectins derived from plants such as, e.g.,concanavalin A, peanut lectin, phytohemagglutinin or wheat-germagglutinin, but they may also comprise purified or enriched physiologicMBL or synthetic, or semi-synthetic mimetics of MBL and/or functionalequivalents of MBL and may be used in an aspect of the inventionrelating to substituting for MBL deficiencies in the complement/lipidpathway. MBL replacement compounds also comprise lipid-C3 conjugates.

In another preferred embodiment, modulators may comprise apoB-replacement factors, which may be functional equivalents of apo Bthat, e.g., exhibit one or more functions of apolipoprotein B48 or B100such as binding to MBL or mimetics thereof and an ability to form aconstituent of a lipoprotein or a mimetic thereof. Such an apoB-replacement factor may be chosen from the group comprising physiologicapo B 48 or B100, natural lipo-oligosaccharides, lipopolysaccharides,lipidated oligo- or polysaccharides, glycoproteins, β-glycyrrhetinicacid, chylomicron-bound sialic acid, phytosterols (β-sitosterol,campesterol, and/or stigmasterol) and (an)other amphiphilic (=partiallyhydrophobic and partially hydrophilic) complement activator(s) (e.g.,mannosylated, N-acetylglucosaminylated, and/or fucosylated phytosterols,or mannosylated, N-acetylglucosaminylated, and/or fucosylated membranelipids, such as phosphoglycerides, glycolipids such as cerebroside organglioside, or sphingomyelin, phosphatidyl choline, phosphatidylserine, phosphatidyl ethanolamine, phosphatidyl inositol, diphosphatidylglycerol or sphingosine), stanols (glycosylated and non-glycosylated),lipidated dextran sulphate(s), (lipo)glucan(s), lipidated tertiary orquaternary ammonium compounds, sialylated glycolipids, combinationsthereof and single and/or combined related substances. In general,suitable apo B-replacement factors comprise amphiphilic compounds orderivatives thereof wherein the hydrophilic part comprises one or morecationic, anionic and/or polar groups and wherein the hydrophobic partcomprises one or more fatty-acid ester moieties. The fatty-acid estermoieties may comprise carbon chain lengths from 1 to 50 carbon atoms,they may be straight and/or branched and they may comprise saturatedand/or unsaturated fatty acids.

Preferred amphiphilic modulators additionally comprise one or more sugarmoieties, such as N-acetylgalactosamine, galactose and/or sialic acid,which allow interaction with a lectin binding site. In a most preferredembodiment according to the invention, such one or more sugar moietiesare mannose, N-acetylglucosamine, and/or fucose moieties that allowinteraction with the lectin binding site of MBL. Other suitable apo Breplacement factors may comprise an IgA or IgD antibody, which isheavily mannosylated, N-acetylglucosaminylated, and/or fucosylated ofeither polyclonal or humanized monoclonal or combinatorial origin,directed towards one of the apolipoproteins of chylomicrons or verylow-density lipoproteins (VLDL). Such antibodies may also be bi-specificantibodies reactive towards both apoB and CR1, thereby being able to,e.g., create bonds between its two antigens.

In another embodiment, modulators may be selected from the groupcomprising MBL and MBL-replacement factors, C4A and functionalequivalents thereof, C4B and functional equivalents thereof, C2 andfunctional equivalents thereof, C3 and functional equivalents thereof,IgG- and IgM-antibodies raised against triglyceride-rich particles andLDL or parts thereof, C3adesArg, factor B and functional equivalentsthereof, factor D and functional equivalents thereof, factor P andfunctional equivalents thereof, serum carboxypeptidases such as sCP-Nand functional equivalents thereof, erythrocyte-bound CR1 and functionalequivalents thereof, free CR1 and functional equivalents thereof, CR1mimetics such as C3b antibodies, vitronectin and functional equivalentsthereof, clusterin and functional equivalents thereof and apo B (48 and100) and apo B replacement factors and esterases such as one of theMASP-proteins and functional equivalents thereof.

In another preferred embodiment, modulators comprise antibodies. In amore preferred embodiment, these antibodies for the classical pathwayare IgG and/or IgM antibodies.

In another embodiment, the group comprising apo B replacement factorsalso comprises an IgA or IgD antibody directed against an apo Blipoprotein, which antibody is heavily mannosylated, and/or heavilyN-acetylglucosaminylated and/or heavily fucosylated.

In a more preferred embodiment, the modulator for the classical pathwayis selected from the group of antibodies wherein the antibody comprisesa polyclonal and/or humanized monoclonal and/or combinatorial antibodyand/or bi-specific antibodies reactive towards both an apo B and CR1.

Administration of a modulator may comprise oral administration, nasaladministration, pulmonary administration, inhalation, anal and/or rectaladministration, intravenous injection, intramuscular injection,intradermal injection, subcutaneous injection, mucous membranediffusion, skin absorption, topical application, extracorporealcirculation-mediated administration and/or any other suitableadministration route, single or in combination.

Modulators may be administered in pure form and/or diluted form, theymay be in solid, semi-solid, crystalline and/or fluidic form, dissolvedand/or dispersed single or as a constituent of a fluid, a spray, a gel,an ointment, a tablet, a suppository, a capsule (synthetic, natural orviral), a powder, a(n) (clinical) intralipid, a (clinical) food product,a (clinical) food additive, a lipidated vaccine for oral application,slow-release and/or direct release carrier that contains the modulatorand/or any other suitable formulation for administration. Furthermore,modulators may be unlabeled or labeled with signal molecules or groupssuch as, e.g., dyes, fluorochromes, radioactive atoms or groups, enzymesor luminescent molecules or groups.

Apo B replacement factors according to the invention may be administeredalone or in combination with other modulators in a natural, artificialor synthetic lipid carrier compound comprising lipoproteins, lipidmicelles, lipid vesicles, artificial lipid bilayer membranes,chylomicrons, liposomes and/or other suitable and/or pharmaceuticallyaccepted lipid substance. Clinical intralipids (fat emulsions) used inrelation to the invention as parenteral feeding may comprise such alipid carrier compound in combination with one or more modulators. In apreferred embodiment of such a parenteral feeding, the lipid carrier isselected from the group comprising mineral oil and natural oils, such assoy oil, sunflower oil, peanut oil, olive oil, palm oil and sesame oiland processed (purified and/or modified) versions thereof. In a mostpreferred embodiment of such a parenteral feeding, the lipid carrier is(purified) olive oil.

It is another embodiment to administer modulators in such a manner thatthe modulator is generated in vivo, e.g., by gene therapy and/or bylocal administration of enzymes (e.g., apo B glycosylation enzymes)their encoding gene(s) and/or gene fragments.

It is a further embodiment to use a method for modulating the activityof one or more elements in the complement/lipid pathway for thetreatment and/or prophylaxis of diseases associated with impairedcomplement-mediated lipid metabolism.

It is a further embodiment to use a method for modulating the activityof one or more elements in the complement/lipid pathway for thetreatment and/or prophylaxis of concomitant (infectious, autoimmune, orneoplastic) diseases that (partially) occupy the lipid eliminatingcomplement activation pathway.

It is a further embodiment to use a method for modulating the activityof one or more elements in the complement/lipid pathway to preventatherogenic processes of concomitant (infectious, autoimmune, orneoplastic) diseases that (partially) occupy the lipid eliminatingcomplement activation pathway.

It is a further embodiment to use a method for modulating the activityof one or more elements in the complement/lipid pathway to efficientlymanipulate the immune system.

It is a further embodiment to use a method for modulating the activityof one or more elements in the complement/lipid pathway to achieveoptimum systemic immunosuppression by lipophilic immunosuppressants.

It is a further embodiment to use a method for modulating the activityof one or more elements in the complement/lipid pathway to achieveoptimum oral immunization. In a preferred such embodiment, a method formodulating the activity of one or more elements in the complement/lipidpathway is used as a lymph-targeting, oro-mucosal adjuvant to induceenhanced mucosal antibody (IgA) responses, T-cell reactivity, and/orsystemic T-cell and/or B-cell (IgM and/or IgG) antibody responses.

It is an embodiment to provide prophylactic measures for diseasesassociated with disturbances in the complement/lipid pathway byproviding improved methods for diagnosing such diseases.

It is one embodiment to estimate the anti-atherogenic potential ofplant-derived, synthetic, or semisynthetic substances by determiningtheir complement activation and/or consumption activity. Complementconsumption should be understood as complement entering the complementcascade thereby disappearing as free component.

It is another embodiment to estimate one or more of the complementcomponents selected from the group comprising MBL, C4A, C4B, C2, factorB, C3adesArg, serum carboxypeptidase N, vitronectin, clusterin, anderythrocyte-bound complement receptor 1 (CR1), in blood, blood serumand/or blood plasma of a patient in order to establish the underlying orrelated defect of his/her atherosclerosis.

It is a further embodiment that concomitant (infectious, autoimmune, orneoplastic) diseases that may (partially) occupy the lipid eliminatingcomplement activation pathway can be diagnosed more adequately so thatatherogenic processes are prevented.

It is another aspect that an individual's lipid profile can bedetermined with greater accuracy by using whole blood rather than bloodplasma in a lipid profile test.

It is a further embodiment to provide compositions for the treatmentand/or prophylaxis of diseases associated with disturbances in thecomplement/lipid pathway. Compositions according to such an embodimentof the present invention may be pharmaceutical compositions, additivesfor pharmaceutical compositions, active substances for pharmaceuticalcompositions, additives for clinical nutrition and/or regular foodadditives and that comprise modulators according to the invention,metabolic precursors of such modulators, biochemically functionalanalogues, functional equivalents and/or derivatives of such modulatorswith or without expedients such as fillers, binders, other complementactivators such as vitamin A, thickening agents, preservatives,lubricants, emulgators, and/or stabilizers.

It is an embodiment that such compositions are used to modulate theactivity of one or more elements of the complement/lipid pathwayaccording to a method of the invention.

EXAMPLES Example 1 Complement Components Associated with Chylomicrons

Experimental procedure: Chylomicrons were isolated from plasma by ultracentrifugation and purified by column chromatography, in the followingmanner: For separation of lipoproteins, plasma samples were subjected toa single ultra-centrifugation step as described in detail.⁽¹²⁾Chylomicron (Sf >1000) and non-chylomicron (Sf <1000) fractions wereseparated by flotation. The chylomicron fraction contained chylomicronsand large VLDL. The non-chylomicron fraction contained chylomicronremnants, small VLDL and its remnants, LDL, HDL and the remainder of theplasma proteins. Aliquots were stored at −20° C. until use. In thefractions containing large chylomicrons (large triglyceride-richparticles) complement components C3, MBL, clusterin (exp. 1), clusterin(exp. 2) and vitronectin were measured by competitive ELISA using thepurified proteins and MBL- and C3-specific polyclonal andclusterin-specific monoclonal-antibodies G7* and EB-8* andvitronectin-specific monoclonal antibody MO-24* as reagents. Thepresence of the complement factors could consistently be demonstrated infractions 13 through 20 (see FIGS. 4A-E). In addition, C3 and MBL werealso found in other lipoproteins isolated by one-step density gradientultra centrifugation (Redgrave gradient) (IDL, LDL, HDL) in subjectsfasting and postprandial after a fat challenge.

Example 2 Adherence of Triglyceride-Rich Particles to Erythrocytes inWhole Blood

Experimental procedure: To observe adherence of triglyceride-richparticles to erythrocytes in whole blood, Sudan Black staining oferythrocytes in whole blood was performed. In this procedure, bloodsmears were prepared and Sudan Black staining was performed with afiltered and saturated solution of Sudan Black in 80% ethanol (4 gramsof Sudan Black B, Electran in 200 ml of 80% ethanol) by the followingprocedure. The blood film on the glass slide was fixed by heat fixation(three times through a flame). The slide was soaked in Sudan Blacksolution for three minutes after which the slide was rinsed with 80%ethanol. The preparation was re-hydrated by a graded ethanol series (oneminute 40% ethanol, one minute 20% ethanol, one minute demineralizedwater). Excess water was shaken off and the slides were dried to air.Microscopic examination of the slides revealed that virtually allerythrocytes of healthy volunteers carried chylomicrons and chylomicronremnants four hours after an oral fat intake (FIG. 5A), whereaserythrocytes in the “fasting” state carried considerably less suchparticles (FIG. 5B).

Example 3 Measurement of Erythrocyte-Bound Apo B-Containing Lipoproteinsby Flow Cytometry

Experimental procedure: Full capillary blood was drawn from non-fastinghealthy subjects by capillary punction. The blood was washed three timesin 10 ml of VSB° buffer (Veronal Saline Buffer) by centrifugation (3,000rpm, ten minutes, 20° C.) and the cell count was adjusted to 1.5×10⁸/mlwith VSB° buffer. A volume of 50 μl of the sample was pelleted and thepellet was re-suspended in 50 μl of a goat raised anti-human apo Bpolyclonal antibody solution (Chemicon 1:25 diluted in VSB° buffer).After a 30-minute incubation of the sample at room temperature (RT), thecells were washed twice in 1 ml of VSB° buffer. The cells were pelletedand resuspended in 50 μl of a FITC-labeled anti-goat antibody solution(Rabbit anti-goat Ig FITC, DAKO 1:10 diluted in VSB° buffer). After a30-minute incubation of the sample at RT, the cells were washed twice in1 ml of VSB° buffer, pelleted, resuspended in 0.5 ml of VSB° buffer andanalyzed by flow cytometry (10,000 cells were counted). Erythrocyteswere gated on forward and side scatter. It could be demonstrated thatthe FITC-label was associated with the side-scattering particles(erythrocytes) only in the presence of the anti-apo B antibodies (FIG.6, bottom panel), whereas no erythrocytes-associated FITC fluorescencecould be detected in the case that incubation with anti-apo B antibodieswas omitted from the analysis (negative control sample, FIG. 6, toppanel). It was therefore concluded that apo B was associated with theerythrocytes in whole blood.

Example 4 Binding and Internalization of Chylomicron Remnants byLeukocytes in the Blood (in Vivo)

Experimental procedure: Fasting venous blood was drawn and Sudan Blackstaining as described in Example 2 was carried out (left panel of FIG.7). In the right panel of FIG. 7, venous blood of the same healthyvolunteer was drawn four hours after administration of a standardizedoral fat load. In the oral RP-fat loading test, cream is used as fatsource; this is a 40% (w/v) fat emulsion with a P/S ratio of 0.06, whichcontains 0.001% (w/v) cholesterol and 2.8% (w/v) carbohydrates. After anovernight fast of 12 hours, the subjects ingest the fresh cream, towhich 120,000 U of aqueous RP (Retinyl palmitate=vitamin A) had beenadded 18 hours before the test, in a dose of 50 g per m² body surface.After the ingestion of the fat load, subjects were only allowed to drinkwater or tea during the following 24 hours. Peripheral blood sampleswere obtained in sodium EDTA (2 mg/ml) before (T=0), at hourly intervalsup to 10 hours and at 12 and 24 hours after the meal. Tubes wereprotected against light by aluminum foil and centrifuged immediately for15 minutes at 800×g at 4° C. Blood samples for FFA measurement werechilled and a lipase inhibitor (Orlistat) was added in order to block invitro lipolysis.

Increased leukocyte concentrations in the postprandial situation areinvolved in the process of atherosclerosis (novel finding by our owngroup).

After having taken up surface fragments from triglyceride-rich particlesor whole remnant particles, neutrophilic granulocytes become activatedand induce a pro-inflammatory response which is the first step in thegeneration of atherosclerosis and endothelial damage.

Example 5 Magnitude and Time-Dependency of Increase of ComplementComponent 3 (C3) in the Postprandial Period

Experimental procedure: Standardized oral fat loading tests (oral RP fatloading test) were performed in volunteers and patients and plasma C3levels were determined nephelometrically at regular intervals.Complement component 3 was measured by nephelometry (Dade BehringNephelometry type II). Maximal postprandial C3 concentrations were inmost cases found after two hours (data not shown). This is consistentwith the concept of chylomicron-driven complement activation (MBLmediated) followed by a compensatory C3 synthesis in vivo. We thereforeconclude that complement activation occurs in vivo during postprandiallipemia (high blood lipid concentrations).

Example 6 Binding and Internalization of Chylomicron Remnants byLeukocytes in the Blood (in Vitro)

Experimental procedure: In vitro incubations of chylomicron remnantswith isolated human leukocytes were performed by methods described inExample 3. Internalization of remnants in leukocytes was observed (datanot shown).

Example 7 Assay for Complement-Activation Cq. Complement-Consumption byDrugs/Food Components Intended for Application in Atherosclerosis orClinical Nutrition

Experimental procedure: In microtiter plates, one “classical” pathwayunit of serum or one “alternative” pathway unit of serum was incubatedfor 0.5 hours with a dilution series of the substance to beinvestigated. In order to do so, the substance of interest is suspendedin micellar form. After incubation, residual classical and alternativecomplement activities are estimated by conventional techniques (J. P. A.M. Klerx, C. J. Beukelman, H. Van Dijk and J. M. N. Willers (1983), J.Immunol. Lett. 63:215-220; H. Van Dijk, P. M. Rademaker and J. M. N.Willers (1985), J. Immunol. Meth. 85: 233-244). The degree of complementconsumption is a measure of complement activation by the components. Alarge number of compounds were identified by this in vitro assay forapplication in atherosclerosis or clinical nutrition.

Example 8 MBL-Dependent Complement Activation by Chylomicrons in HumanSerum

Experimental procedure: Chylomicrons were isolated from human serum byultra centrifugation and purified by column chromatography. The purifiedchylomicron fractions were added to MBL-positive serum (from healthyhuman subjects) and MBL-negative serum (from MBL-deficient humansubjects) and purified heterologous chicken erythrocytes were added.Complement activation was allowed to occur at 37° C. for 45 minutesafter which the extent of hemolysis was evaluated byspectrophotometrical determination of hemoglobin levels in serumsupernatants. It was found that hemolysis of heterologous erythrocyteswas extensive in the case that an MBL-positive serum was used, whereashemolysis was virtually absent in the case of an MBL-negative serum(data not shown). This demonstrated that chylomicrons can bring aboutcomplement activation in human serum in an MBL-dependent manner.

Using the MBL in-vitro assay, we identified the components from oliveoil and soy oil inducing Complement Lipid Pathway (CliP)-activation.

The results are summarized in FIG. 9.

From these results, two compounds were selected which show strongCLiP-induction and which are known to be safe to be administered tohuman (thanks to other clinical use), specifically (i) glycosylatedplant sterols and (ii) vitamin A.

Example 9 Postprandial C3 Buildup

Experimental procedure: Full capillary blood was draw from healthysubjects and the C3 levels were determined together with the leukocytecount. Postprandial (situation in blood after a meal) leukocyte increaseand activation was associated with postprandial complement C3 increase.In the early postprandial phase (<4 hours) predominantly neutrophilicgranulocytes were observed, whereas between four and ten hours into thepostprandial period, an increase of lymphocytes was observed. Thesefindings were consistent with the notion that leukocytes play a role inatherosclerosis by the formation of foam cells.

Example 10 Effect of Glycosylated Plant Sterols on Fasting PlasmaTriglycerides and Cholesterol Levels in Two MBL-Deficient Patients andin One MBL-Normal Patient with Heterozygous FamilialHypercholesterolemia

Proof of principle has been reached in two MBL-deficient patients.

These subjects were treated with a diet enriched in glycosylated plantsterols during three weeks. The glycosylated plant sterols were selectedin the MBL in vitro test as described in Example 8. This interventionresulted in a decrease of fasting plasma triglycerides and cholesterol(Table 1).

TABLE 1 Effect of glycosylated plant sterols on blood parameters ofMBL-deficient patients. Plasma TG (mM) Cholesterol (mM) apoB (g/L)Before After Before After Before After MBL def1 3.66 2.27 4.8 4.2 0.850.89 MBL def2 0.88 0.79 4.8 3.6 0.62 0.56

Using a different intervention with glycosylated plant stanols in apatient with heterozygous Familial Hypercholesterolemia (with relativelynormal MBL activity in plasma), refractory to therapy with expanded dosestatins in combination with a lipid lowering diet and resins,significant reductions of plasma cholesterol (from 10 to 7.8 mmol/L),fasting plasma triglycerides (from 2.3 to 1.08 mmol/L) and plasma apoB(from 1.90 tot 1.62 g/L) were achieved reaching the lowest concentrationever experienced by this patient (FIG. 10). This example provides invivo support for the Complement Lipid Pathway (CliP) concept developedby C-Tres, using a sub-optimal lead.

Example 11 Effect of Vitamin a on Post-Prandial CliP Stimulation

Another series of lead compounds, namely vitamin A-analogues, weretested in 20 healthy volunteers in order to determine the CLiPstimulating potency of these leads that had shown CLiP stimulation invitro (Example 8). Twenty healthy volunteers were tested on twodifferent occasions. Blood was drawn before and after ingestion of astandardized oral fat load with and without vitamin A (as representativefor these series of leads) given to the participants in random order.Addition of vitamin A to the oral fat load resulted in a significantlyhigher postprandial plasma C3 increase, whereas the same amount of fatwas ingested in both situations (FIG. 11 a).

The levels of plasma trygliceride increase two hours after acute oralfat load also showed a reduction in the volunteers if the fat load wasgiven with vitamin A (FIG. 11 b).

This is in line with the CLiP concept that by activating the Complementsystem, plasma triglycerides will be reduced even in healthynormolipidemic subjects. Note: it should be stressed that the C3increase in this group of young, healthy, lean subjects was expected tobe lower due to the characteristics of the subjects. In older,insulin-insensitive subjects the postprandial C3 response is muchhigher.

These experiments in human gave the expected results uponadministration: increase of C3-titers and decrease of triglycerides. Itis therefore reasonable to assume that also other compounds, active inthe in vitro assay, show CLiP-activities in human.

Other Analytical Methods:

Triglyceride-rich particles in plasma, chylomicrons and non-chylomicronswere determined by HPLC as described⁽¹⁴⁾ TG and cholesterol weremeasured in duplicate by commercial calorimetric assay (GPO-PAP, andMonotest Cholesterol kit, Boehringer Mannheim) as described.^((14, 23))Plasma apo B and apo AI were determined by immunoturbidimetry.⁽²³⁾ Apo Egenotype was determined as described.⁽⁴⁷⁻⁴⁹⁾ HDL2 and HDL3 cholesterolconcentrations were determined by precipitation procedures asdescribed.⁽⁵⁰⁾ Complement factor 3 was measured immunoturbidimetry ornephelometrically. Acylation stimulating protein was determined byELISA, as were factor B and D. Ketone bodies were measured by HPLC.

REFERENCES

-   1. Ross R. The pathogenesis of atherosclerosis: a perspective for    the 1990's. Nature 1993; 362:801-809.-   2. Willeit J., S. Kiechl, F. Oberhollenzer, G. Rungger, G. Egger, E.    Bonora, M. Mitterer, and M. Muggeo. Distinct risk profiles of early    and advanced atheroclerosis. Prospective results from the Brunneck    Study. Arterioscl. Thromb. Vasc. Biol. 2000; 20:529-537.-   3. Bucher H. C., L. E. Griffith, and G. H. Guyatt. Systematic review    on the risk and benefit of different cholesterol-lowering    interventions. Arterioscl. Thromb. Vasc. Biol. 1999; 19:187-195.-   4. The Heart Outcomes prevention Evaluation Study Investigators.    Effects of an angiotensin-converting-enzyme inhibitor, ramipril, on    cardiovascular events in high-risk patients. N. Engl. J. Med. 2000;    342:145-53.-   5. The Diabetes Control and Complication Trial Research Group. The    effect of intensive treatment of diabetes on the development and    progression of long-term complications in insulin-dependent diabetes    mellitus. N. Engl. J. Med. 1993; 329:977-86.-   6. UK Prospective Diabetes Study (UKPDS) Group. Intensive    blood-glucose control with sulphonylureas or insulin compared with    conventional treatment and risk of complications in patients with    type 2 diabetes (UKPDS 33). Lancet 1998; 352:837-53.-   7. Haffner S. M., S. Lehto, T. Ronnemaa, K. Pyorala, and M. Laakso.    Mortality from coronary heart disease in subjects with type 2    diabetes and in nondiabetic subjects with and without prior    myocardial infarction. N. Engl. J. Med. 1998; 339:229-34.-   8. Ross R. Atherosclerosis: an inflammatory disease. N. Engl. J.    Med. 1999; 340:115-126.-   9. Law S. K. A. and K. B. M. Reid: Complement in focus, 2nd edition,    1995, Oxford University Press, Oxford, G.B.-   10. Rosenberg M. E. and J. Silkensen: Clusterin: Physiologic and    pathophysiologic considerations. Int. J. Biochem. Cell Biol. 1995;    27(7):633-645.-   11. Ishikawa Y., Y. Akasaka, T. Ishii, K. Komiyama, S. Masuda, N.    Asuwa, N.-H. Choi-Miura, and M. Tomita: Distribution and synthesis    of apolipoprotein J in the atherosclerotic aorta. Artherioscler.    Thromb. Vasc. Biol. 1998; 18:665-672.-   12. Madsen H. O., V. Videm, A. Svejgaard, J. L. Svennevig, and P.    Garred: Association of mannose-binding-lectin deficiency with severe    atherosclerosis. The Lancet 1998; 352:959-960.

1. A method for the treatment and/or prophylaxis of diseases associatedwith disturbances in the complement/lipid pathway, said methodcomprising: modulating the activity of one or more elements in thecomplement/lipid pathway of a subject.
 2. The method according to claim1, wherein the activity of one or more elements of a lectin pathway, aclassical pathway and/or an alternative pathway for complementactivation are modulated.
 3. The method according to claim 1, whereinthe disease is atherogenic.
 4. The method according to claim 1, whereinthe disease is atherosclerosis and/or an underlying and/or relateddisease.
 5. The method according to claim 1, wherein said modulating theactivity of one or more elements is achieved through administering oneor more modulators to the subject.
 6. The method according to claim 5,wherein the modulator is selected from the group consisting of MBL andMBL-replacement factors, C4A, C4B, C2, C3, IgG- and IgM-antibodiesraised against triglyceride-rich particles and LDL or parts thereof,C3adesArg, factor B, factor D, factor P, serum carboxypeptidases, sCP-N,erythrocyte-bound CR1, free CR1, CR1 mimetics, C3b antibodies,vitronectin, clusterin, and apo B (48 and 100) and apo B replacementfactors, esterases, an MASP-protein, and a functional equivalent ormixture of any thereof.
 7. The method according to claim 5, wherein themodulator is selected from the group consisting of MBL-replacementfactors and apo B replacement factors.
 8. The method according to claim1, wherein said modulator is an antibody.
 9. The method according toclaim 8, where said antibody is selected from the group consisting ofIgG antibodies, IgM antibodies, and mixtures thereof.
 10. The methodaccording to claim 6, wherein the modulator comprises apo B replacementfactors and a heavily mannosylated IgA or IgD antibody directed againstan apo B lipoprotein.
 11. The method according to claim 6, wherein themodulator comprises apo B replacement factors and a heavilyN-acetylglucosaminylated IgA or IgD antibody directed against an apo Blipoprotein.
 12. The method according to claim 6, wherein the modulatorcomprises apo B replacement factors and a heavily fucosylated IgA or IgDantibody directed against an apo B lipoprotein.
 13. The method accordingto claim 8, wherein said antibody is selected from the group consistingof polyclonal antibodies, humanized monoclonal antibodies, combinatorialantibody, and mixtures thereof.
 14. The method according to claim 8,wherein said antibody comprises bi-specific antibodies reactive towardsboth an apo B and CR1.
 15. The method according to claim 5, wherein themodulator is administered via parenteral feeding in an intralipidcarrier.
 16. The method according to claim 15, wherein the intralipidcarrier is olive oil.
 17. The method according to claim 5, wherein themodulator is generated in vivo in the subject.
 18. The method accordingto claim 1 for the treatment and/or prophylaxis of diseases selectedfrom the group consisting of diseases associated with impairedcomplement-dependent lipid metabolism, atherosclerosis and/or underlyingand/or related diseases, and atherogenic processes of concomitant(infectious, autoimmune, or neoplastic) diseases that at least partiallyoccupy the lipid eliminating complement activation pathway.
 19. A methodfor diagnosing disturbances in the complement/lipid pathway or anunderlying or related defect of atherosclerosis, said method comprising:determining the presence and/or abundance of at least one element of thecomplement/lipid pathway in a sample.
 20. (canceled)
 21. The methodaccording to claim 19, wherein said at least one element of thecomplement/lipid pathway is selected from the group consisting of MBL,C4A, C4B, C2, factor B, factor D, C3adesArg, serum carboxypeptidase N,vitronectin, clusterin, chylomicron-bound sialic acid, anderythrocyte-bound complement receptor 1 (CR1).
 22. The method accordingto claim 21, wherein additionally at least one concomitant (infectious,autoimmune, or neoplastic) disease that may at least partially occupythe lipid eliminating complement activation pathway is diagnosed. 23.The method according to claim 21, wherein additionally a subject's lipidprofile is determined by using whole blood.
 24. The method according toclaim 21, for discovering pharmaceutical and/or nutritional compoundsfor the treatment and/or prophylaxis of atherogenic disturbances oflipid metabolism related to disturbances in the complement/lipidpathway.
 25. (canceled)
 26. A composition for the treatment and/orprophylaxis of diseases selected from the group consisting of diseasesassociated with disturbances in the complement/lipid pathway,atherosclerosis and/or an underlying and/or a related disease associatedwith disturbances in the complement/lipid pathway, and disturbances oflipid metabolism, said composition comprising: at least one modulator ofthe complement/lipid pathway.
 27. The composition of claim 26, whereinsaid composition modulates the activity of one or more elements of thelectin pathway and/or the alternative pathway for complement activation.28. The composition of claim 26, wherein said modulator is selected fromthe group consisting of MBL and MBL-replacement factors, C4A, C4B, C2,C3, IgG- and IgM-antibodies raised against triglyceride-rich particlesand LDL or parts thereof, C3adesArg, factor B, factor D, factor P, serumcarboxypeptidases, sCP-N, erythrocyte-bound CR1, free CR1, CR1 mimetics,C3b antibodies, vitronectin, clusterin, apo B (48 and 100) and apo Breplacement factors, esterases, an MASP-protein, and functionalequivalents and mixtures of any thereof.
 29. The composition of claim26, wherein said modulator is selected from the group consisting ofMBL-replacement factors and apo B replacement factors.
 30. Thecomposition of claim 26, wherein said modulators are metabolicprecursors of modulators.
 31. The composition of claim 26, furthercomprising a pharmaceutically acceptable carrier selected from the groupconsisting of natural lipid carriers, artificial lipid carriers,synthetic lipid carriers, mineral oil, natural oil, processed mineraloil, natural oil, and mixtures thereof.
 32. A kit for diagnosingatherogenic disturbances of lipid metabolism or diagnosingatherosclerosis by a method according to any of the claims 18, said kitcomprising: means for receiving a sample, and means for carrying out anassay for the detection of at least one modulator and/or element of thecomplement/lipid pathway in the sample.